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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 May 2010 to 08 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted to current internationally accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
Time interval between the third reference material flask and the first test material solution flask was more than 15 minutes. This deviation was presumed to exert no effect on the study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
yes
Remarks:
Time interval between the third reference material flask and the first test material solution flask was more than 15 minutes. This deviation was presumed to exert no effect on the study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
yes
Remarks:
Time interval between the third reference material flask and the first test material solution flask was more than 15 minutes. This deviation was presumed to exert no effect on the study
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A desired amount of test item was directly weighed into the designated test flasks for each nominal concentration. The test material concentrations were prepared by mechanical dispersion using an ultrasonic bath for 5 minutes. The test solutions were freshly prepared at the begining of the experiment in the testing laboratory.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Preparation of inoculum for exposure: The activated sludge was obtained from a domestic sewage plant in Veszprem, Hungary. The activated sludge was firstly washed, then centrifuged. The supernatant liquid phase was decanted. The soil material was re-suspended in isotonic saline solution and again centrifuged. The was repeated twice. An aliquot of the final sludge suspension was weighed and dried, and the ratiio of wet sludge to dry weight determined. Based on this ratio, the calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (dry weight). The pH of the activated sludge inoculum was determined to be pH 7.45. The activated sludge was used directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
Approximately 10 minutes.
Test temperature:
19.8-20.7 °C (during incubation) and 20.2-21.0 °C (during oxygen measurement)
pH:
7.16-7.24
Dissolved oxygen:
From 7.9 to 8.2 mg O2/L
Nominal and measured concentrations:
10, 31, 100, 313 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 350 mL Erlenmeyer bottles and BOD bottles with a special 300 mL neck.
- Aeration: Compressed air (1 litre per minute)
- Amount of activated sludge in test vessels: 132 mL (4 g/L)
- Test conditions: Test vessels were held in a climate chamber (during the incubation period) and a controlled environment room (during the formulation and respiration measuring)

TEST MEDIUM / WATER PARAMETERS
Synthetic Sewage Feed (ratio of composition of culture media referring to 1000 mL)
Peptone 16.0 g
Meat extract: 11.0 g
Urea 3.0 g
NaCl 0.7 g
CaCl2 x 2H2O: 0.4 g
MgSO4 x 7H2O: 0.2 g
K2HPO4 : 2.8 g
Deionised water add 1000.0 mL


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in all treatments. The temperature was measured in the climate chamber with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all BOD bottles.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorphenol
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
95.58 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: 95% CI 69.36-131.71
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
614.67 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: 95% CI 420.87-879.73
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
3 952.95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: 1995.03-7832.35
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Results with reference substance (positive control):
- Results with reference substance valid?The 3-hour EC50 of the reference item 3,5-Dichlorophenol for the used activated sludge batch was determined to be in the range of 5 to 30 mg/L and therefore considered valid.
- Relevant effect levels:The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 6.94 mg/L with 95% confidence limits of 4.53 to 10.64 mg/L.
Reported statistics and error estimates:
The inhibitory effect of the test material and the reference material at a particular concentration on the respiration rate of the sludge were expressed as a percentage of the mean value for the respiration rate of the two controls using the following equation:

% inhibition = [1- ((2Rs/(Rc1 + Rc2))] x 100
Where:
Rs = Oxygen consumption rate at tested concentration of test item
Rc1 = oxygen consumption rate of control 1
Rc2 = oxygen consumption rate of control 2

The 3-hour EC20, EC50 and EC80 of the test item and the 3-hour EC50 of the reference item with 95% confidence limits were calculated by Probit analysis using TOXSTAT software.

Table 2: Influence of test item on oxygen consumption of activated sludge

Flask No.

ID

Test group

Concentration (mg/L)

Oxygen consumption (mg O2/L/min)

Inhibition (%)

pH-values

Oxygen concentration (mg O2/L)

Start*

End*

Start*

End*

1

C1

Control

-

0.590

-

7.21

7.26

8.2

7.7

15

C2

Control

-

0.570

-

7.24

7.37

7.9

7.3

 

Mean

-

0.580

-

-

-

-

-

Deviation

-

3.45

-

-

-

-

-

10

T1

Test material

10

0.550

5.2

7.20

7.49

7.9

8.0

11

T2

Test material

31

0.550

5.2

7.16

7.47

7.9

7.9

12

T3

Test material

100

0.430

25.9

7.18

7.61

7.9

7.5

13

T4

Test material

313

0.390

32.8

7.19

7.60

7.9

7.4

14

T5

Test material

1000

0.220

62.1

7.19

7.66

7.9

7.9

* start and end of 3-hour aeration

ID code of each group

 

Table 3: Influence of test material of oxygen consumption of activated sludge

Concentration (mg/L)

Number subject

Number observed

Observed proportion

Predicted proportion

10

100

5

0.0500

0.0313

31

100

5

0.0500

0.0884

100

100

25

0.2500

0.2058

313

100

32

0.3200

0.3801

1000

100

62

0.6200

0.5871

Chi2lack of fit = 6.1632

Table Chi2= 7.8147, alpha = 0.05, df = 3

Likelihood lack of fit = 6.2836

 

Table 4: Influence of reference material on oxygen consumption of activated sludge

Flask No.

ID

Test group

Concentration (mg/L)

Oxygen consumption (mg O2/L/min)

Inhibition (%)

pH va;ues

(Oxygen concentration (mg O2/L)

Start*

end

Start*

End*

1

C1

Control

-

0.590

-

7.21

7.26

8.2

7.7

15

C2

Control

-

0.570

-

7.24

7.37

7.9

7.3

 

Mean

-

0.580

-

-

-

-

-

Deviation

-

3.45

-

-

-

-

-

2

R1

Reference material

5

0.320

44.8

7.23

7.82

8.2

7.8

3

R2

Reference material

16

0.200

65.5

7.34

7.52

8.1

8.0

4

R3

Reference material

32

0.140

75.9

7.31

7.65

8.0

8.3

* start and end of 3-hour aeration

ID code of each group

 

Table 5: Influence of reference material on oxygen consumption of activated sludge

Concentration [mg/L]

Number subject

Number observed

Observed proportion

Predicted proportion

5

100

44

0.4400

0.4417

16

100

65

0.6500

0.6455

32

100

75

0.7500

0.7527

Chi2lack of fit = 0.0139

Table Chi2= 3.8415, alpha = 0.05, df = 1

Likelihood lack of fit = 0.0139

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 8.6% and 91.4% in the examined nominal test concentration range (10-1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested. Based on the measured inhibition rates for 3 hours, the EC20, EC50 and EC80 and their 95% confidence limits were calculated to be:

EC20:                     95.58 mg/L (69.36-131.71)

EC50:                     614.67 mg/L (420.87-897.73)

EC80:                     3952.95 mg/L (1995.03-7832.35)

The NOEC was determined to be 31 mg/L

The NOEC for respiration rates was not derived through statistical analysis, but is biologically justified. The respiration rates were inhibited and influence dose dependently in the whole concentration range, and the observed slight inhibition (5.2%) at the concentration level of 31 mg/L was considered to be due to biological variability in the test. The calculated respiration rate , 0.550 (at 31 mg/L) was in the historical control data range (0.521 ± 0.071), and the deviation from the control was within ± 15%, this can therefore be considered as biological variability of the test system.

At the nominal concentration of 16 and 32 mg/L of the reference material, the respiration rate was inhibited by 65.5% and 75.9% respectively. The 3-hour EC50of 3,5-dichlorophenol was calculated to be 6.94 mg/L with 95% confidence limits of 4.53 to 10.64 mg/L.

Validity criteria fulfilled:
yes
Remarks:
Comments regarding validity criteria are included under the section overall remarks
Conclusions:
In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 8.6% and 91.4% in the examined nominal test concentration range (10-1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested. Based on the measured inhibition rates for 3 hours, the EC20, EC50 and EC80 and their 95% confidence limits were calculated to be:
EC20:                     95.58 mg/L (69.36-131.71)
EC50:                     614.67 mg/L (420.87-897.73)
EC80:                     3952.95 mg/L (1995.03-7832.35)
The NOEC was determined to be 31 mg/L
The NOEC for respiration rates was not derived through statistical analysis, but is biologically justified. The respiration rates were inhibited and influence dose dependently in the whole concentration range, and the observed slight inhibition (5.2%) at the concentration level of 31 mg/L was considered to be due to biological variability in the test. The calculated respiration rate , 0.550 (at 31 mg/L) was in the historical control data range (0.521 ± 0.071), and the deviation from the control was within ± 15%, this can therefore be considered as biological variability of the test system.
At the nominal concentration of 16 and 32 mg/L of the reference material, the respiration rate was inhibited by 65.5% and 75.9% respectively. The 3-hour EC50of 3,5-dichlorophenol was calculated to be 6.94 mg/L with 95% confidence limits of 4.53 to 10.64 mg/L.
Executive summary:

In a GLP study conducted to current internationally accepted guidelines, Sipos (2010) investigated the potential of Great Lakes DBS to inhibit the growth of activated sludge. The test material was tested at concentration of 0, 10, 31, 100, 313 and 1000 mg/L, a reference material was also run alongside the main experiment with 3,5 -dichlorophenol at concentrations of 16 and 32 mg/L. The respiration rate was measured my a stirring O2electrode and was recorded for about ten minutes. The oxygen consumption (in mg O2L-1minute-1) was determined from the most linear part of the respiration curve.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 8.6% and 91.4% in the examined nominal test concentration range (10-1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested. Based on the measured inhibition rates for 3 hours, the EC20, EC50 and EC80 and their 95% confidence limits were calculated to be:

EC20:                     95.58 mg/L (69.36-131.71)

EC50:                     614.67 mg/L (420.87-897.73)

EC80:                     3952.95 mg/L (1995.03-7832.35)

The NOEC was determined to be 31 mg/L

Description of key information

The inhibition of growth (measured via oxygen consumption) of activated sludge by the test material (Benzene, ethenyl-, ar - bromo derivs) was studied in the report by Sipos (2010). The study was performed to current internationally accepted guideline and was GLP compliant. The study was considered to be of high reliability and as such suitable for use to fulfil the data requirement as a stand alone study

Key value for chemical safety assessment

EC50 for microorganisms:
614.67 mg/L
EC10 or NOEC for microorganisms:
31 mg/L

Additional information

Reference: Sipos K (2010)

Reliabilty: 1

Results: In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 8.6% and 91.4% in the examined nominal test concentration range (10-1000 mg/L). Concentrations exceeding 1000 mg/L nominal were not tested. Based on the measured inhibition rates for 3 hours, the EC20, EC50 and EC80 and their 95% confidence limits were calculated to be:

EC20:                     95.58 mg/L (69.36-131.71)

EC50:                     614.67 mg/L (420.87-897.73)

EC80:                     3952.95 mg/L (1995.03-7832.35)

The NOEC was determined to be 31 mg/L